The intrinsic tenase complex (ITC) is rate-limiting for thrombin generation, and animal models suggest that therapeutic targeting of this complex reduces bleeding risk. Our central hypothesis is that the heparin-binding exosite of factor IXa is critical to regulation of intrinsic tenase activity, thrombin generation, and response to injury. This hypothesis is based on our demonstration that heparin inhibits the ITC by disrupting the interaction of factor VIlla with this exosite. The rationale for these studies is that compounds utilizing this mechanism will represent effective antithrombotic agents with reduced bleeding risk. The specific aims of this proposal are to: 1) determine the extent to which the factor VIlla and heparin-binding sites overlap on factor IXa, 2) demonstrate the role of the heparin binding exosite in the ex vivo and in vivo regulation of factor IXa activity, and 3) identify novel compounds that specifically target this exosite. Recombinant factor IX mutants will be expressed, purified, and characterized with respect to factor IXa-heparin affinity by surface plasmon resonance and factor IXa-factor VIlla affinity by functional binding assays. The mutant proteins will be critical reagents to analyze the contribution of this exosite to ex vivo coagulant activity, half-life, and thrombin generation in the absence and presence of heparins. Likewise, the effect of these mutations on plasma clearance, cellular degradation by the LDL receptor-related protein (LRP), response to venous endothelial injury, and efficacy of heparins will be examined in the hemophilia B mouse. Finally, glycosaminoglycans and small molecule chemical libraries will undergo high throughput screening for compounds that disrupt the factor IXa-heparin interaction, and these compounds will be characterized with regard to ITC inhibition. These studies will provide a detailed molecular understanding of how heparin disrupts critical protease interactions with the factor VIlla A2 domain, demonstrate the critical role of the factor IXa heparin-binding exosite for thrombin generation, in vivo clearance of factor IX(a) and response to injury, and the therapeutic efficacy of heparins. Finally, these studies will provide proof of principle for this exosite as a critical therapeutic target, and identify lead compounds for a novel class of antithrombotics.